Autocatalytic replication of recombinant RNA

ABSTRACT

This invention concerns recombinant RNA molecules comprising a recognition sequence for the binding of an RNA-directed RNA polymerase, a sequence for the initiation of product strand synthesis and a heterologous sequence of interest inserted at a specific site in the internal region of the recombinant molecule. Such recombinant RNA molecules are capable of serving as a template for the synthesis of complementary single-stranded molecules by RNA-directed RNA polymerase. The product molecules so formed are also capable of serving as a template for the synthesis of additional copies of the original recombinant RNA molecule. In a preferred embodiment of the invention Qβ replicase is used as the RNA-directed RNA polymerase.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referenced by thenames of the authors and the year of the publication within parentheses.Full citations for these publications may be found at the end of thespecification immediately preceding the claims. The disclosures of thesepublications in their entireties are hereby incorporated by referenceinto this application in order to more fully describe the state of theart to which this invention pertains.

The synthesis of RNA in vitro by Qβ replicase (Haruna & Spiegelman,1965a) is remarkable because a small number of template strands caninitiate the synthesis of a large number of product strands (Haruna &Spiegelman, 1965b). A 100,000-fold increase in RNA can occur during aten-minute reaction (Kramer et al., 1974). This striking amplificationis the consequence of an autocatalytic reaction mechanism (Spiegelman etal., 1968; Weissmann et al., 1968). Single-stranded RNAs serve astemplates for the synthesis of complementary single-stranded products.Both the product strand and the template strand are released from thereplication complex and are free to serve as templates in subsequentrounds of synthesis (Dobkin et al., 1979). Consequently, the number ofRNA strands increases exponentially as the reaction proceeds.

Many investigators have attempted to exploit the autocatalytic nature ofQβ replicase reactions in order to synthesize large amounts of any RNAin vitro. However, Qβ replicase does not copy most RNAs. Like otherviral RNA-directed RNA polymerases it is highly selective for its owntemplate (Haruna & Spiegelman 1965c). In vivo this enables Qβ replicaseto distinguish bacteriophage Qβ RNA from the vast number of differentRNA molecules that are present in Escherichia coli. This templatespecificity is a consequence of two separate interactions that occurbetween the replicase and Qβ RNA. First, the replicase binds strongly toa unique internal recognition sequence (Weber et al., 1974; Vollenweideret al., 1976; Meyer et al., 1981). Then, product strand synthesis isinitiated at a cytidine-rich sequence located at the 3' end of thetemplate (Rensing & August, 1969; Schwyzer et al., 1972). Each of thesesequences must be present in both complementary strands forautocatalytic synthesis to occur. A number of strategies have beendevised to circumvent these barriers to the synthesis of heterologousRNAs by Qβ replicase. Manganese was used to decrease the stringency ofthe interactions between the replicase and the template (Palmenberg &Kaesberg, 1974; Obinata et al., 1975); primers were used to bypass thenormal initiation step (Feix & Hake, 1975; Feix, 1976; Vournakis et al.,1976); and polycytidine tails were added to templates to mimic therequired 3'-terminal sequence (Kuppers & Sumper, 1975; Owens & Diener,1977). These strategies were tried with a wide range of heterologoustemplates, including rRNAs, viral RNAs and eukaryotic mRNAs. In allcases, the amount of RNA synthesized never exceeded the original amountof template RNA and the products consisted only of complementarystrands. Consequently, synthesis was not autocatalytic and these methodscould not approach the efficiency with which Qβ RNA is synthesized by Qβreplicase.

In a different strategy a poly (A) molecule was inserted between two QβRNA molecules which had been partially degraded, one from the 3'-end andanother from the 5' end. E. coli HrH photoplasts were infected withthese RNA molecules and two phage clones carrying poly (A) in their RNAwere obtained after reproduction of the phage in vivo. (Tongjian andMeiyan, 1982). The yield of recombinant RNA molecules produced by thismethod was very low and its infectivity was between 1/1000 and 1/10000that of the wild type RNA.

This invention concerns a method for the autocatalytic synthesis ofheterologous RNAs in vitro by Qβ replicase. Our approach was toconstruct a recombinant RNA by inserting a heterologous sequence into anatural Qβ replicase template. The template we used, MDV-1 (+) RNA(Kacian et al., 1972), is only 221 nucleotides long, and its completenucleotide sequence (Mills et al., 1973; Kramer & Mills, 1978) andsecondary structure (Mills et al., 1980) have been determined. Themechanism of its replication by Qβ replicase has been studied in detail(Mills et al., 1978; Dobkin et al., 1979; Kramer & Mills, 1981; Bauschet al., 1983) and has been shown to be fundamentally similar to thereplication of Qβ RNA. In particular, it possesses a highly structuredinternal binding site for Qβ replicase (Nishihara et al., 1983) and acytidine-rich 3'-terminal sequence that is required for product strandinitiation (Mills et al., 1980). We selected an insertion site at aposition where the heterologous sequence would not interfere with thesefunctional regions and where it would not disturb the structure of theMDV-1 RNA. We hoped that the replicase would respond to the recombinantRNA as it would to a natural template.

The recombinant RNA was constructed by cleaving MDV-1 (+) RNA at theselected site and then inserting decaadenylic acid in that site with theaid of bacteriophage T4 RNA ligase. This recombinant RNA was then usedas a template in a reaction containing Qβ replicase. The productconsisted of full-length copies of the recombinant RNA. Furthermore,both complementary strands were synthesized. The reaction proceededautocatalytically, resulting in an exponential increase in the amount ofrecombinant RNA.

SUMMARY OF THE INVENTION

A recombinant single-stranded RNA molecule comprising a recognitionsequence for the binding of an RNA-directed RNA polymerase, a sequencefor the initiation of product strand synthesis by the polymerase and aheterologous sequence of interest derived from a different RNA moleculeinserted at a specific site in the internal region of the recombinantmolecule, has been synthesized.

In a specific embodiment of the invention this recombinant RNA moleculeis capable of serving as a template for the synthesis of a complementarysingle-stranded RNA molecule by an RNA-directed RNA polymerase. Theproduct of this synthesis is also capable of serving as a template forthe synthesis of additional copies of the original recombinant RNAmolecule by the polymerase.

In specific embodiments of the invention the recognition sequence forthe binding of an RNA directed RNA polymerase is in the internal regionof the molecule.

The specific insertion site for the heterologous RNA sequence ofinterest which is derived from a different molecule is not near thebinding sequence of the polymerase or the sequence for the initiation ofproduct strand synthesis. In a preferred embodiment of the invention theinsertion site is at a specific nucleotide in a region where theinserted sequence has a minimal effect upon the structure of thetemplate RNA molecule.

In a preferred embodiment of the invention the RNA template molecule ismidivariant RNA (MDV-1 RNA) which serves as a template for Qβ replicase.A specific heterologous RNA sequence of interest is inserted at a sitewhere the insertion would not significantly effect the replicability ofthe resulting combinant, e.g. between nucleotides 63 and 64 of MDV-1 (+)RNA.

The present invention also concerns a novel method of cleaving an RNAmolecule at a specific site. According to the invention a modified cDNAmolecule is hybridized to the RNA molecule to be cleaved. Thenon-complementary loop of the heteroduplex is cleaved by a ribonucleaseat a specific nucleotide to yield the desired fragments. The inventionalso concerns methods of constructing recombinant RNA molecules fromsuch fragrants and methods of synthesizing such recombinant RNAmolecules in vitro.

Recombinant RNAs constructed by this method are useful as hybridizationprobes, since they can be highly labeled during synthesis, and sinceblotting with RNA instead of DNA results in lower backgrounds.Recombinants can also be made from unprocessed gene transcripts toprovide a ready source of substrates for the isolation of processingenzymes and for use in studies probing RNA splicing mechanisms.Recombinants can also be made from eucaryotic messenger RNAs that aredifficult to obtain. They can serve as a virtually unlimited source ofmRNAs for use as templates in cell-free translation systems. Thus,recombinant mRNAs may provide a novel means for obtaining usefulquantities of rare proteins. Recombinant RNAs also provide a means fordirectly sequencing the heterologous insert by replication in thepresence of 3'-deoxyribonucleoside 5'-triphosphate chain terminators.Furthermore, recombinant RNAs can be constructed from the genomes ofviruses and viroids. Mutants of these recombinants can then be selectedthrough the use of in vitro evolution techniques that have beendeveloped for Qβ RNA. The mutant heterologous sequences can be recoveredfrom the recombinants and can then be introduced into cells to studytheir altered biological activity. In summary, the autocatalyticreplication of recombinant ribonucleic acids constructed from genomesand gene transcripts provides a powerful tool for probing andmanipulating genetic information.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Secondary structure of MDV-1 (+) RNA, showing the site at whichdecaadenylic acid was inserted into the sequence (indicated by anarrow). Bold letters identify those nucleotides that are found inhomologous regions of Qβ (-) RNA (Nishihara et al., 1983). Of 46nucleotides between positions 81 and 126, 40 are identical withnucleotides 84 to 129 of Qβ (-) RNA and 30 of 35 nucleotides betweenpositions 187 and 221 are identical with nucleotides 4186 to 4220 at the3' end of Qβ (-) RNA. These regions contain major elements of theinternal replicase binding site and the product strand initiation site.

FIG. 2. Heteroduplex formed by the hybridization of MDV-1 (+) RNA tomodified MDV-1 (-) cDNA. The arrow indicates the site at whichribonuclease T₁ cleaved the RNA strand into 2 fragments. Sequencehyphens have been omitted for clarity.

FIG. 3. Construction of a recombinant RNA: (1) a phosphate was added tothe 5' end of the 158-nucleotide fragment of MDV-1 (+) RNA; (2)decaadenylic acid (lacking terminal phosphates) was ligated to thephosphorylated 158-nucleotide fragment, forming a 168-nucleotidefragment: (3) a phosphate was added to the 5' end of the 168-nucleotidefragment: (4) the 63-nucleotide fragment of MDV-1 (+) RNA wasdephosphorylated; and (5) the dephosphorylated 63-nucleotide fragmentwas ligated to the phosphorylated 168-nucleotide fragment, forming a231-nucleotide recombinant RNA.

FIG. 4. Comparison of the kinetics of MDV-1 RNA synthesis andrecombinant RNA synthesis. The logarithm of the amount of RNA present ineach sample was plotted as a function of incubation time. The amount ofRNA in each reaction increased exponentially, until the number of RNAstrands equaled the number of active replicase molecules, and thereafterincreased linearly.

DETAILED DESCRIPTION OF THE INVENTION

A recombinant single-stranded RNA molecule comprising a recognitionsequence for the binding of an RNA-directed RNA polymerase, a sequencefor the initiation of product strand synthesis by the polymerase and aheterologous sequence of interest derived from a different RNA moleculeinserted at a specific site in the internal region of the recombinantmolecule has been synthesized.

The recombinant RNA molecule is capable of serving as a template invitro for the synthesis of a complmentary single-stranded RNA moleculeby an RNA-directed RNA-polymerase. This complementary product moleculeis also capable of serving as a template in vitro for the synthesis ofadditional copies of the original recombinant molecule by theRNA-directed RNA polymerase.

The recognition sequence on the recombinant molecule for the binding ofthe RNA-directed RNA-polymerase is located in an internal region of themolecule. In preferred embodiments of the invention the insertion sitefor the heterologous RNA sequence of interest is not located near anysurface required for the binding of the RNA polymerase or for theinitiation of product strand synthesis, in order not to interfere withthe template activity of the recombinant RNA molecule. It is alsodesirable to locate this insert in a position in the molecule were itseffect upon the secondary structure of the molecule, and thus itstemplate activity, will be minimal.

In a specific embodiment of the invention the sequence required for theinitiation of product strand synthesis is a cytidine rich sequencelocated on the 3' terminal end of the molecule.

In another embodiment, the recombinant RNA molecule contains at leastone radiolabeled nucleotide. In a preferred embodiment, the nucleotidecontains numerous radiolabeled nucleotides.

In a specific embodiment of the invention the RNA-directed RNApolymerase is Qβ replicase. The recombinant RNA molecules in thisembodiment contain a template for Qβ replicase and a heterologous RNAsequence of interest derived from a different RNA molecule inserted, asin other embodiments, at a specific site in the template, e.g., at ornear a specific nucleotide.

The Qβ replicase template can be variant RNA, e.g., midivariant RNA,minivariant RNA, microvariant RNA, one of the nanovariant RNAs, othervariants to which names have not yet been assigned, or mutants thereof.It is preferred that the insertion site of the heterologous RNA sequenceof interest be located in a region where the sequences are not known tobe required for replication, e.g., in a loop where viable mutations areknown to occur, and is preferably on the exterior of the molecules,e.g., at or near a guanosine residue which is hypersusceptible tocleavage by ribonuclease T₁.

In a specific embodiment of the invention the template is MDV-1 RNA or amutant thereof. Either the MDV-1 (+) RNA or MDV-1 (-) RNA molecules maybe used.

In a presently preferred embodiment the heterologous RNA sequence ofinterest is inserted, e.g., between nucleotides 63 and 64 of the MDV-1(+) RNA molecule. The heterologous RNA sequence may be, e.g. a naturalor synthetic mRNA, a primary gene transcript or a transcript obtained byin vitro transcription from recombinant DNA or genomic RNA. In aspecific embodiment of the invention, the heterologous sequence isdecaadenylic acid and it is inserted between nucleotides 63 and 64 ofthe MDV-1 (+) RNA molecule.

The invention also concerns single-stranded RNA and DNA molecules whichare complementary to the single-stranded RNA template. Thesecomplementary molecules can be prepared by any conventional method, e.g.by enzymatic or chemical synthesis.

In one embodiment, a molecule complementary to the Qβ replicase templatemolecule may be prepared. An MDV-1 (-) cDNA molecule was thus preparedenzymatically by reverse transcriptase from MDV-1 (+) RNA. This moleculeand other cDNA molecules of this invention can be cloned as an insert ina DNA-vector such as a plasmid, e.g. pBR322.

Another aspect of this invention concerns modified single-stranded cDNAmolecules useful in a method for cleaving an RNA molecule at a specificsite. In one embodiment, the modified cDNA molecule is modified in thata segment of the DNA molecule is replaced with a non-homologous segment,of DNA. The DNA segment replaced is the segment of the cDNA moleculewhich is complementary to the nucleotide sequence of a desired specificcleavage site on the RNA molecule. The non-homologous replacementsegment is not capable of hybridizing to the nucleotides of the specificcleavage site. The non-homologous replacement may contain a differentnumber of nucleotides than the RNA cleavage site. In one embodiment theDNA replacement segment contains eight nucleotides and the RNA cleavagesite contains three nucleotides. This non-homologous segment can be thecleavage site of a restriction enzyme, e.g. endonuclease XbaI. Themodifications may be performed enzymatically or chemically. In this andother embodiments the RNA may be a template for an RNA-directed RNApolymerase, e.g., Qβ replicase. Where the polymerase is Qβ replicase thetemplate may be midivariant RNA.

In a specific embodiment of the invention a modified cDNA molecule issubstantially complementary to MDV-1 (+) RNA and contains a modificationin the region complementary to the specific cleavage site of the MDV-1(+) RNA between nucleotides 63 and 64. The MDV-1 (-) cDNA modificationcomprises the replacement the 3 base pair DNA segment complementary toRNA nucleotides 62 to 64 with an 8 nucleotide non-homologous segment. Inone embodiment the non-homologous segment is C--T--C--T--A--G--A--G,which contains the XbaI site.

Modified cDNA molecules prepared according to this invention may beobtained in large quantities by inserting them into a vector DNAmolecule, e.g. plasmid pBR322, and producing them in large quantities inhost cells, e.g. E. coli.

The modified cDNA molecules so produced are useful in methods ofcleaving a single-stranded RNA molecule at specific sites. A modifedcDNA molecule substantially complementary to the RNA molecule to becleaved as described above, e.g. an MDV-1 (+) RNA molecule and asubstantially complementary MDV-1 (-) cDNA molecule, is incubated withthe RNA molecule to be cleaved under suitable conditions and for asufficient period of time permitting the hybridization of substantiallycomplementary molecules. The heteroduplex formed is isolated uponcompletion of hybridization and contains a non-hybridized loop at theregion where the strands are not complementary. The RNA strand of theheteroduplex is then cleaved in the region of the non-complementaryloop, e.g. between nucleotides 63 and 64 of MDV-1 (+) RNA. Site-specificcleavage of the RNA may be effected by a variety of means includingchemical and enzymatic reactions. Presently preferred, however, is theuse of a ribonuclease, e.g. ribonuclease T₁ to cleave the RNA. Thecleaved heteroduplex may be isolated and separated, e.g. by melting toobtain single-stranded RNA and DNA molecules. The RNA fragments soobtained are separated and purified, to yield in one embodiment usingribonuclease T₁, a 63 nucleotide MDV-1 (+) RNA fragment with a natural5' terminal triphosphate and a 158 nucleotide MDV-1 (+) RNA fragmentwith a natural 3' terminal hydroxyl group.

Fragments of RNA molecules which are useful as templates for thesynthesis of RNA by an RNA-directed RNA polymerase can be obtained bythese specific cleavage methods. Since these fragments have been cleavedat a specific known cleavage site they are useful as components ofrecombinant RNA molecules.

The present invention also concerns methods for the construction ofrecombinant RNA molecules from the RNA template fragments obtained bysite specific cleavage. A 5' terminal phosphate is added to the fragmentthat acquired a 5' terminal hydroxyl group as a result of the cleavageby ribonuclease T₁, e.g. the 158 nucleotide MDV-1 (+) RNA fragment. Aheterologous RNA sequence of interest derived from a different molecule,e.g. decaadenylic acid, is ligated to the 5' terminus of thephosphorylated fragment. The ligation can be by chemical or enzymaticmethods, but it is preferably performed enzymatically with bacteriophageT₄ RNA ligase. After the ligation is completed a 5' terminal phosphateis added to the ligated product, e.g. the 168 nucleotide MDV-1 (+) RNAfragment containing decaadenylic acid. The terminal phosphates are thenremoved from the fragment which acquired a 3' terminal phosphate as aresult of the cleavage by ribonuclease T.sub. 1, e.g. the 63 nucleotideMDV-1 (+) RNA fragment. This dephosphorylated fragment is then ligatedto the product of the previous ligation and the resulting recombinantRNA molecule is isolated and purified.

In a preferred embodiment of the invention MDV-1 (+) RNA is used as thevector for the construction of a recombinant RNA molecule. The use ofthis natural Qβ replicase template, should allow for the introduction oflarge heterologous RNA sequences into the recombinant molecules. Thisshould be possible since the natural substrate for Qβ replicase, Qβ RNA,is approximately 4200 nucleotides in length, whereas MDV-1 RNA is 221nucleotides in length. Any type of heterologous single-stranded RNAmolecule, from any origin may be an insert in the recombinant RNAmolecules of this invention.

The invention also concerns in vitro methods of synthesizing therecombinant RNA molecules so constructed. The recombinant RNA moleculesare incubated with the RNA-directed RNA polymerase for which they serveas a template under suitable conditions permitting RNA replication.Large amounts of the recombinant RNA molecules can thus be obtained.

In a specific embodiment of the invention MDV-1 (+) recombinant RNAcontaining an insert between nucleotides 63 and 64 is incubated with Qβreplicase. These recombinant molecules are capable of autocatalyticreplication wherein the complementary product strand produced alsoserves as a template for synthesis of the recombinant RNA the Qβreplicase.

Another embodiment of the invention concerns a method of specificallycleaving the MDV-1 (+) RNA recombinant molecule which contains thedecaadenylic sequence. Cleavage by this method results in fragments ofthe MDV-1 (+) RNA molecule in which the phosphate and hydroxyl residuesare in correct orientation for the ligation of another heterologous RNAsequence of interest. The recombinant MDV-1 (+) RNA molecule containingthe decaadenylic acid sequence is incubated with decathymidylic acidunder suitable conditions permitting the hybridization of complementarystrands. The hybrid so formed is treated with ribonuclease H undersuitable conditions permitting the digestion of the decaadenylateinsert. The RNA fragments obtained are then separated and isolated. Thismethod of cleavage results in an MDV-1 (+) RNA fragment of about 63nucleotides containing a 3' terminal hydroxyl group and an MDV-1 (+) RNAfragment of about 158 nucleotides containing a 5' terminal phosphategroup. These fragments can then be used in a ligation reaction withother heterologous RNA sequences of interest to construct other MDV-1(+) RNA recombinant molecules capable of autocatalytic replication.

In a specific embodiment of the invention the recombinant RNA moleculescan be used to sequence a specific nucleic acid sequence of interest.The nucleic acid sequence of interest is present as a heterologous RNAinsert at a specific site of the recombinant molecule. The recombinantmolecule is autocatalytically replicated in vitro by Qβ replicase in thepresence of 3' deoxyribonucleoside 5'-triphosphate chain terminators.The RNA molecules of various length so obtained are then analyzed bystandard molecular biology sequencing techniques.

Recombinant RNA molecules constructed from appropriate sequences arealso useful as hybridization probes in a method for determining thepresence or concentration of an oligo- or polynucleotide, e.g. DNA, ofinterest in a mixture. Suitable recombinant RNA molecules may beobtained by preparing an RNA molecule complementary to the oligo- orpolynucleotide of interest by methods known in the art. Using thecomplementary RNA strand so obtained as a heterologous segment of arecombinant RNA molecule of this invention permits the convenientautocatalytic synthesis of recombinant RNA molecules useful ashybridization probes for the material of interest. Recombinant RNAmolecules of this invention are particularly advantageous in suchmethods because they can be densely labeled with radiolabelednucleotides and since blotting with RNA instead of DNA results in lowerbackgrounds. According to one method of this invention a radiolabeledrecombinant RNA molecule or derivative thereof is contacted with amixture under suitable conditions and for a sufficient period of timepermitting complementary nucleotide segments to hybridize. Therecombinant RNA molecule or fragment thereof contains a nucleotidesegment complementary to the oligo- or polynucleotide of interest. Thepresence or intensity of radioactivity in hybridized nucleotide segmentsis then determined and correlated with the presence or concentration ofthe oligo- or polynucleotide of interest.

This method is widely applicable for use in any of the availableconfigurations useful for DNA hybridization probes. It may be used in adiagnostic device or kit for the detection of nucleotide segmentsassociated with a disorder in plants or animals, including humans.

In another embodiment of the invention a recombinant RNA molecule may beused as a hybridization label in a method for labeling an oligo- orpolynucleotide of interest in biological material, e.g., a tissuesample. The recombinant RNA or a derivative thereof contains at leastone, and preferably more than one, radiolabeled nucleotide. The RNA orRNA fragment also contains a nucleotide segment complementary to theoligo- or polynucleotide of interest. The method involves contacting theradiolabeled RNA or RNA derivative with the biological material undersuitable conditions and for a sufficient period of time to permitcomplementary nucleotide segments to hybridize. Hybridization accordingto this method effectively radiolabels the oligo- or polynucleotide ofinterest.

In a further embodiment, the recombinant RNA molecules or derivativesthereof may be used in a cell-free method of protein synthesis. Therecombinant RNA useful in this method is prepared by the methoddisclosed above and contains a heterologous nucleotide sequence codingfor a desired protein. The RNA itself may be used in this method.Alternatively, the heterologous sequence may be cleaved from thetemplate molecule after replication and used in place of the completerecombinant molecule. In either case, the heterologous sequence may alsocontain additional nucleotide sequences, e.g., one or more introns, a 5'cap or polyadenylic acid tail, and may be further derivatized, e.g.methylated. In accordance with this method, a mixture is preparedcontaining the RNA or RNA-derivative coding for the desired protein, acellular extract capable of synthesizing a protein encoded by therecombinant RNA or derivative, and an effective amount of appropriateamino acids and buffer. The cellular extract may be obtained frombacterial cells, or from eucaryotic cells, e.g. rabbit reticulocytes orwheat germ, and contains ribosomes and various soluble enzymes. Themixture is incubated under suitable conditions permitting enzymaticsynthesis of the encoded protein. By this method rare proteins andproteins of significant scientific, medical or commercial utility may beconveniently obtained in small scale reactions as well as on anindustrial scale.

In another embodiment, the recombinant RNA molecules of this inventionmay be used in a method for identifying or characterizing an RNAprocessing enzyme in a biological material. According to this method arecombinant RNA molecule is prepared by the methods disclosed above andcontains a heterologous sequence comprising a primary gene transcript.The primary gene transcript is a substrate for the RNA processing enzymeof interest. The RNA molecule or a derivative thereof which alsocomprises a substrate for the enzyme of interest is incubated with thebiological material under suitable conditions permitting an RNAprocessing enzyme to cleave a substrate RNA molecule. By detecting thepresence of character of RNA cleavage products it is possible toidentify or characterize the enzyme. In a specific embodiment, the RNAmolecule is a transcript of a recombinant DNA molecule, the transcriptcomprising a substrate of the enzyme of interest.

In a further embodiment, a recombinant RNA molecule of this inventionmay be used in a method for producing a mutant virus or viroid. Therecombinant RNA molecule contains a heterologous inserted sequencecomprising the RNA genomes of the virus or viroid of interest.Incubating the recombinant molecule under appropriate selectiveconditions and for a sufficient period of time permits the accumulation,i.e., the selection, of a population of mutant replicates. Suitableselective conditions are numerous and varied, but include replicatingthe recombinant molecules in the presence of a chain elongationinhibitor, e.g. ethidium bromide; a nuclease, e.g., ribonuclease T₁ ; ora chain terminator, e.g., cordycepin 5'-triphosphate. Mutant viruses orviroids so obtained may be useful in exploring the role of differentnucleotide sequences in pathogenicity, infectivity and replicability.Furthermore, mutants of reduced pathogenicity may be obtained by thismethod which may be protect plant or animal from infection by a morepathogenic form.

In still another embodiment of this invention the recombinant RNAmolecules may be used in a method for isolating one type of RNA from amixture of different RNA molecules. The method involves preparing amixture of recombinant RNA molecules according to the methods describedherein. The heterologous fragments incorporated into the template RNAmolecules to form the recombinant molecules comprise the mixture of RNAmolecules containing the RNA of interest. The mixture of recombinant RNAmolecules is serially diluted until convenient aliquots may be withdrawnfrom the mixture, each of which containing one molecule of recombinanton average. Each aliquot is then separately incubated with anappropriate RNA-directed RNA polymerase under suitable conditionspermitting autocatalytic synthesis of copies of the recombinant RNA.Homogeneous populations of recombinant copies are then identified. Thehomogeneous populations so identified are screened for populationscontaining copies of the recombinant molecule containing the RNA ofinterest.

The following experimental procedures are set forth to illustratespecific embodiments of the invention. While these embodiments utilizemidivariant RNA, this invention contemplates the use of other RNAtemplates for other RNA-directed RNA polymerases. Furthermore, theinvention contemplates the use of any heterologous RNA segment which maybe inserted in such RNA templates at various specific template sites.

MATERIALS AND METHODS

(a) Materials

Qβ replicase was isolated from bacteriophage Qβ-infected E. coli Q13,using the procedure of Eoyang & August (1971) with thehydroxylapatite-chromatography step omitted. The following enzymes werepurchased: bacteriophage T₄ RNA ligase from P-L Biochemicals, Milwaukee,WI; bacteriophage T₄ polynucleotide kinase from Boehringer-MannheimBiochemicals, Indianapolis, IN; bacterial alkaline phosphates andrestriction endonuclease EcoRI from Bethesda Research Laboratories,Bethesda, Md.; proteinase K from EM laboratories, Elmsford, N.Y.; andribonuclease T₁ from Calbiochem, La Jolla, Calif. Decaadenylic acid(lacking terminal phosphates) was obtained from P-L Biochemicals.Radioactive ribonucleoside 5'-triphosphates were purchased from ICN,Irvine, Calif., and unlabeled ribonucleoside 5'-triphosphates wereobtained from P-L Biochemicals.

(b) MDV-1 (+) RNA

The synthesis of MDV-1 RNA (Kramer et al., 1974) and the isolation ofthe complementary (+) and (-) strands by polyacrylamide slab gelelectrophoresis in the presence of magnesium ions (Mills et al., 1978)have been described. A mutant MDV-1 RNA was used in these studies. Itsnucleotide sequence differed from the wild-type sequence at position 61of the (+) strand, where there was an adenosine in place of a guanosineresidue (Kramer et al., unpublished results).

(c) Modified MDV-1 cDNA

The synthesis of MDV-1 cDNA by avian myeloblastosis virus reversetranscriptase, its cloning into the EcoRI site of pBR322 DNA, and themodification of its nucleotide sequence in the region of its uniqueHinfI site have been described (Bausch et al., 1983). The modificationinvolved the replacement of a 3 base-pair segment (nucleotides 62 to 64in the MDV-1 (+) strand and nucleotides 157 to 159 in the (-) strand)with an 8 base-pair segment containing an XbaI site(C--T--C--T--A--G--A--G in each strand). Modified MDV-1 cDNA wasisolated from the plasmid by digestion with endonuclease EcoRI.

(d) Site-directed cleavage of MDV-1 (+) RNA

MDV-1 (+) RNA was hybridized to MDV-1 (-) cDNA in an aqueous formamidesolution (Casey & Davidson, 1977) as follows: 34 μg of [α-³²P]GTP-labeled MDV-1 (+) RNA and 68 μg of modified MDV-1 cDNA wereincubated in 50 μl of 800 mg. formamide/ml, 300 mM NaCl, 30 mM sodiumcitrate (pH 6.5) at 80° C. for 10 min to melt the double-stranded DNA.This solution was then incubated at 55° C. for 24 h to promoteannealing. Then 100 μl of standard buffer (20 mM Tris-HCl (pH 7.5), 400mM NaCl, 3mM EDTA, 1 mg sodium dodecyl sulfate/ml) was added, and theMDV-1 (+) RNA/MDV-1 (-) cDNA heteroduplex was isolated from the mixtureby gel filtration chromatography on Sepharose 4B eluted in standardbuffer. The hybrid was then precipitated with 2 vol. ethanol.

The MDV-1 (+) RNA in the heteroduplex was cleaved between nucleotides 63and 64 by incubating 48 μg of the hybrid at 0° C. for 60 min in 3 ml of100 units of ribonuclease T₁ /ml, 100 mM Tris-HC1 (pH 7.4), 20 mM MgCl₂.The volume was brought to 6 ml with the addition of proteinase K to 50μg/ml, sodium dodecyl sulfate to 5 mg/ml, Tris-HCl (pH 7.4) to 100 mM,NaCl to 400 mM, and EDTA to 30 mM. This solution was incubated at 23° C.for 60 min to destroy the ribonuclease. Protein was then extracted twicewith an equal volume of water-saturated phenol and the nickedheteroduplex was precipitated with 2 vol. ethanol. The cleavedheteroduplex was melted apart in 7M urea at 100° C., and the RNAfragments were separated electrophoretically (Mills & Kramer, 1979) andeluted from the gel. The length and identity of each fragment wasconfirmed by nucleotide sequence analysis (Sanger et al., 1965).

(e) Construction of the recombinant RNA

A 5'-terminal phosphate was added to the 158-nucleotide fragment of thecleaved MDV-1 (+) RNA according to the method of Donis-Keller et al.(1977): 6 μg of the fragment were incubated at 50° C. for 3 min in 140μl of 1mM spermidine, 10 mM Tris-HCl (pH 7.4), 100 M EDTA, and thenrapidly chilled to 0° C. The volume was brought to 200 μl with theaddition of bacteriophage T₄ polynucleotide kinase to 225 units/ml,[γ-³² P]adenosine 5'-triphosphate to 3 μM, Tris-HCl (pH 7.4) to 50 mM,MgCl₂ to 10 mM, and dithiothreitol to 5 mM. This solution was incubatedat 37° C. for 2 h. The reaction was terminated by the addition of 200 μlof standard buffer. Protein was extracted with an equal volume of phenoland the phosphorylated fragment was precipitated with 2 vol. ethanol.

Decaadenylic acid (lacking terminal phosphates) was ligated to the 5'end of the phosphorylated 158-nucleotide fragment as follows: thedecaadenylic acid, which was purchased as an ammonium salt, wasconverted to a sodium salt by dissolving it in 400 mM NaCl andprecipitating it with 2 vol. ethanol. Sodium decaadenylic acid (50 nmol)and 4 μg of phosphorylated 158-nucleotide fragment were incubated at 50°C. for 3 min in 70 μl of 1 mM spermidine, 10 mM Tris-HCl (pH 7.4), 100μM EDTA, and then rapidly chilled to 0° C. The volume was brought to 100μl with the addition of bacteriophage T₄ RNA ligase to 400 units/ml,Tris-HCl (pH 7.4) to 50 mM, MgCl₂ to 10 mM, and dithiothreitol to 5 mM.This solution was incubated at 4° C. for 30 h. The reaction wasterminated by the addition of 100 μl of standard buffer. Protein wasextracted with an equal volume of water-saturated phenol and the RNA wasprecipitated with 2 vol. ethanol. The RNA was then separated bypolyacrylamide gel electrophoresis and the 168-nucleotide ligationproduct was eluted from the gel.

A 5'-terminal phosphate was added to the 168-nucleotide ligation productas follows: 1 μg of the 168-nucleotide product was incubated at 37° C.for 2 h in 20 μl of 225 units T₄ polynucleotide kinase/ml, 2 μM-[γ-³²P]adenosine 5'-triphosphate, 50 mM Tris--HCl (pH 7.4), 10 mM MgCl₂, 5 mMdithiothreitol. The reaction was terminated by the addition of 80 μl ofstandard buffer. Protein was extracted with an equal volume ofwater-saturated phenol and the RNA was precipitated with 2 vol. ethanol.The phosphorylated 168-nucleotide product was then purified further bypolyacrylamide gel electrophoresis.

The terminal phosphates of the 63-nucleotide fragment of the cleavedMDV-1 (+) RNA were removed as follows: 2 μg of the fragment wereincubated at 37° C. for 2 h in 100 μl of 14 units of bacterial alkalinephosphatase/μl, 100 mM Tris-HCl (pH 8.3), 20 mM MgCl₂. The reaction wasterminated by the addition of 100 l of 100 mM EDTA, 800 mM NaCl, 10 mgsodium dodecyl sulfate/ml. Protein was extracted with an equal volume ofwater-saturated phenol and the dephosphorylated fragment wasprecipitated with 2 vol. ethanol.

The dephosphorylated 63-nucleotide fragment was ligated to the 5' end ofthe phosphorylated 168-nucleotide product as follows: 2 μg of thedephosphorylated 63-nucleotide fragment and 100 ng of the phosphorylated168-nucleotide product were incubated at 37° C. for 2 h in 10 μl of 400units of T₄ RNA ligase/ml, 50 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, mMdithiothreitol. The reaction volume was then doubled by the addition offresh buffer and ligase, and incubation was continued at 4° C. for 48 h.The reaction was terminated by the addition of 80 μl of standard buffer.Protein was extracted with an equal volume of water-saturated phenol andthe RNA was precipitated with 2 vol. ethanol. The RNA was then separatedby polyacrylamide gel electrophoresis: 310 pg of the 231-nucleotiderecombinant RNA were recovered from the gel.

(f) Replication of the recombinant RNA

The synthesis of recombinant RNA by Qβ replicase was carried outaccording to the protocol of Kramer et al. (1974). Recombinant templateRNA (100 pg) was incubated for 15 min in a 12.5 μl reaction volume. Theproduct RNA was electrophoretically purified, melted in 7M urea at 100°C. and the complementary (+) and (-) strands were separated (Mills etal., 1978). The identity of each of the complementary recombinant RNAstrands was confirmed by nucleotide sequence analysis.

(g) Kinetic analysis of recombinant RNA synthesis

Recombinant (-) RNA (392 pg) was incubated at 37° C. in 75 μl of 20 μgQβ replicase/ml, 84 mM Tris--HCl (pH 7.4), 12 mM MgCl₂, 200 μM ATP, 200μM CTP, 200 μM UTP and 250 μM [α-³² P]GTP. A control reaction was alsoprepared, in which 375 pg of MDV-1 (-) RNA was used as template. Samples(5 μl) were taken from each reaction at 1 min intervals and immediatelydiluted with 200 μl of standard buffer containing 10 μg of unlabeledyeast RNA as carrier. The protein in each sample was extracted with anequal volume of phenol and the RNA in 100 μl of each aqueous phase wasprecipitated with 2 vol. ethanol. The RNA samples were then analyzed, inparallel, by polyacrylamide gel electrophoresis. Their homogeneity wasconfirmed by an examination of the autoradiograph. The amount of RNA ineach gel band was determined by scintillation counting. These data wereused to compare the rate of recombinant RNA synthesis with the rate ofMDV-1 RNA synthesis.

RESULTS

(a) Selection of the insertion site

Autocatalytic replication of a recombinant RNA cannot occur if theaddition of the heterologous sequence to the Qβ replicase templateinterferes with normal template function. We therefore chose aninsertion site in MDV-1 (+) RNA that was not near any sequence known tobe required for replication. In addition, the selected insertion sitewas in a region where the presence of a heterologous sequence would havea minimal effect on template structure. FIG. 1 shows the secondarystructure of MDV-1 (+) RNA predicated by computer analysis (Zuker &Stiegler, 1981) and established by chemical modification (Mills et al.,1980), observation of band compression regions in sequencing gels(Kramer & Mills, 1978; Mills & Kramer, 1979), and structure mapping withribonuclease T₁ (Kramer et al., 1981). Two regions of MDV-1 (+) RNA thatare homologous with regions of Qβ (-) RNA are shown in the Figure. Oneis the highly structured internal region that contains major elements ofthe template recognition site (Nishihara et al., 1983). The other is thecytidine-rich 3'-terminal sequence required for product strandinitiation (Mills et al., 1980). The insertion site that we selected waslocated away from these regions, between nucleotides 63 and 64.

There are additional reasons why this particular site was chosen.Computer analysis indicated that the ends of the fragments generated bycleavage at the site were likely to be single-stranded, which wouldfacilitate their subsequent ligation (England & Uhlenbeck, 1978). Thesite was located in a hairpin loop, where it was least likely to disturbthe structure. Viable mutations were known to occur in this hairpin loop(Kramer et al., unpublished results), suggesting that its sequence wasnot essential for replication. And finally, the site was probably on theexterior of the molecule, because the guanosine residue in the hairpinloop is hypersusceptible to cleavage by ribonuclease T₁ (Kramer et al.,unpublished results).

(b) Site-directed cleavage

To cleave MDV-1 (+) RNA between nucleotides 63 and 64, a heteroduplexwas formed between [α-³² P]GTP-labeled MDV-1 (+) RNA and modified MDV-1(-) cDNA. The modified MDV-1 (-) cDNA contained an eight-nucleotidesequence, C--T--C--T--A--G--A--G, in place of the sequence that iscomplementary to nucleotides 62 to 64 of the (+) strand (Bausch et al.,1983). Consequently, the RNA in the heteroduplex was completelyhybridized to the DNA except in the region of the modified sequence (seeFIG. 2). The heteroduplex was then digested with ribonuclease T₁. Sinceribonuclease T₁ cleave RNA only on the 3' side of guanosine residues insingle-stranded regions (Sato-Asano, 1959), the modified cDNA served asa mask, limiting cleavage to the 3' phosphodiester bond of the loneexposed guanosine residue at position 63. The cleaved heteroduplex wasisolated from the digestion mixture, melted apart, and the two RNAfragments were separated from each other by polyacrylamide gelelectrophoresis. Each fragment was eluted from the gel and its lengthand homogeneity were confirmed by nucleotide sequence analysis. The63-nucleotide fragment possessed a natural 5'-terminal triphosphate andacquired a 3'-terminal phosphate as a consequence of cleavage. The158-nucleotide fragment acquired a 5'-terminal hydroxyl group as aconsequence of the cleavage and possessed a natural 3'-terminal hydroxylgroup.

(c) Construction of the recombinant RNA

The insertion of decaadenylic acid between the two MDV-1 (+) RNAfragments was accomplished in two stages with the aid of bacteriophageT₄ RNA ligase (Silber et al., 1972), utilizing methods developed byKaufmann & Littauer (1974) and Uhlenbeck & Cameron (1977). First, thedecaadenylic acid was ligated to the 5' end of the 158-nucleotidefragment, forming a 168-nucleotide fragment. Then, the 63-nucleotidefragment was ligated to the 5' end of the 168-nucleotide fragment,forming a 231-nucleotide recombinant RNA. Since T₄ RNA ligase requires a3'-hydroxyl group on the fragment containing the reactive 3' end and a5' phosphate on the fragment containing the reactive 5' end (Kaufmann &Kallenbach, 1975; Walker et al., 1975), preliminary reactions werecarried out to convert the ends of each fragment to the appropriateform. FIG. 3 summarizes the steps required to construct the recombinantRNA.

The 5' end of the 158-nucleotide fragment was phosphorylated bybacteriophage T₄ polynucleotide kinase, in a reaction that utilized[α-³² P]ATP. Decaadenylic acid, which lacked terminal phosphates, wasthen added to the 5' end of the phosphorylated 158-nucleotide fragmentwith the aid of T₄ RNA ligase. There were 624 times as many moles ofdecaadenylic acid present in the reaction as there were moles of158-nucleotide fragment. The presence of a large excess of thenon-phosphorylated fragment increased the rate of ligation and minimizedthe undesirable dimerization of the phosphorylated fragment (Walker etal., 1975). The 168-nucleotide ligation product was isolated bypolyacrylamide gel electrophoresis. Analysis of the gel indicated that60% of the 158-nucleotide fragments had been ligated to decaadenylicacid. Each synthetic step was monitored by nucleotide sequence analysis.Before phosphorylation, the 5' end of the 158-nucleotide fragmentyielded a unique oligonucleotide, U--C--A--C--G. After the addition of a5'-terminal phosphate (of a much higher specific radioactivity than wasused to label the internal phosphates), the 5' end of the 158-nucleotidefragment was seen as pU--C--A--C--G, which migrates to a differentposition in the fingerprint pattern that U--C--A--C--G. The ligation ofdecaadenylic acid to the 5' end of the phosphorylated 158-nucleotidefragment resulted in the disappearance of pU--C--A--C--G and theappearance of a highly labeled oligonucleotide that migrated to aposition in the fingerprint pattern (Sanger et al., 1965) thatidentified it as the expected (A)₁₀ U--C--A--C--G.

In preparation for the next ligation, the 5' end of the 168-nucleotideligation product was phosphorylated by T₄ polynucleotide kinase in areaction that utilized [γ-³² P]ATP (also at a much higher specificradioactivity than was used to label the internal phosphates), and the63-nucleotide fragment was dephosphorylated by bacterial alkalinephosphatase. There were 50 times as many moles of dephosphorylated63-nucleotide fragment present in the reaction as there were moles ofphosphorylated 168-nucleotide fragment. After ligation, the RNA wasisolated and analyzed by polyacrylamide gel electrophoresis. A band thatmigrated slightly slower than a MDV-1 (+) RNA marker in the adjacentlane was seen in the autoradiograph of the gel. The RNA in this band wasrecovered from the gel and a portion was examined by nucleotide sequenceanalysis. Two oligonucleotides were seen in the fingerprint pattern. Oneoligonucleotide was in the position expected for (A)₁₀ U--C--A--C--G,which derived its radioactivity from the phosphate that had been addedto the 5' end of the 158-nucleotide fragment. The other oligonucleotidewas in the position expected for A--C--A--A--G, which was the3'-terminal oligonucleotide of the 63-nucleotide fragment, and whichcould have derived its radioactivity only from the 5'-terminal phosphatethat had been added to the 168-nucleotide fragment. Thus, the RNA wasthe expected 231-nucleotide recombinant formed by the insertion ofdecaadenylic acid between A--C--A--A--G and U--C--A--C--G in thesequence of MDV-1 (+) RNA: 0.2% of the 168-nucleotide fragments had beenligated to the 63-nucleotide fragment.

(d) Autocatalytic synthesis of the recombinant RNA

The isolated recombinant RNA was used as a template for Qβ replicase.The products were isolated from the reaction and were analyzed bypolyacrylamide gel electrophoresis. Two sizes of RNA were seen. Thesmaller species comigrated with MDV-1 RNA. The larger RNA, whichrepresented 70% of the total, was thus identified as recombinant RNA.Each RNA was eluted from the gel and its complementary (+) and (-)strands were melted apart in 7M urea and isolated by polyacrylamide gelelectrophoresis. Each complementary strand of the recombinant RNAmigrated more slowly than the corresponding strand of MDV-1 RNA. Thepresence of both (+) and (-) strands in the recombinant RNA productindicated that its synthesis was autocatalytic. The probable source ofthe MDV-1 RNA in the Qβ replicase reaction was that some 158-nucleotidefragments had contaminated the 168-nucleotide fragments used during thesecond ligation, resulting in the presence of MDV-1 (+) RNA in therecombinant RNA template. Subsequenct Qβ replicase reactions wereinitiated with pure recombinant RNA isolated from the strand-separationgel, and the products of these reactions were not contaminated withMDV-1 RNA.

Fingerprint patterns of strand-separated recombinant RNA were comparedwith those of MDV-1 RNA to confirm its identity. The only differenceseen with [α-³² P]GTP-labeled (+) strands was that U--C--A--C--G, whichwas present in MDV-1 (+) RNA, was replaced by (A)₁₀ U--C--A--C--G in therecombinant (+) RNA. In the complementary (-) strands, labeled with[α-³² P]UTP to emphasize the heterologous sequence, the only differenceseen was that A--C--U--U--G, which was present in MDV-1 (-) RNA, wasreplaced in the recombinant (-) RNA by a large oligonucleotide that wasrich in uridine. Measurement of the radioactivity of theoligonucleotides present in the (-) strand fingerprint patternsindicated that the A--C--U--U--G (whose 3' phosphate was derived from anearest-neighbor uridine) contained radioactive phosphates from 3 of 33uridine residues present in MDV-1 (-) RNA, and the large oligonucleotidecontained radioactive phosphates from 13 of 43 uridine residues presentin the recombinant (-) RNA. The large oligonucleotide of the recombinant(-) RNA was therefore A(U)₁₀ C--U--U--G, which is complementary to theheterologous sequence inserted into MDV-1 (+) RNA.

The kinetics of recombinant RNA synthesis were determined by observingtwo separate Qβ replicase reactions, one employing recombinant RNA astemplate, and the other employing MDV-1 RNA as template. Before theinitiation of synthesis, there was a great excess of Qβ replicasecompared with template RNA. Samples were taken from each reaction at oneminute intervals and the amount of RNA in each was determined. Theamount of RNA present in each reaction increased exponentially with time(see FIG. 4). The rate at which the population of recombinant RNAmolecules increased in number was comparable to the rate of increase ofthe MDV-1 RNA population. Within nine minutes, the number of recombinantRNA molecules increased 300-fold. Thus, the insertion of a heterologoussequence within MDV-1 RNA did not interfere with replication; themechanism of recombinant RNA synthesis was autocatalytic; and the amountof recombinant RNA increased exponentially.

Discussion

The 231-nucleotide recombinant RNA will be useful as a vehicle for thereplication of other RNAs in vitro. In preliminary experiments, we haveshown that the recombinant RNA, which contains decaadenylic acid, can becleaved into two fragments by hybridizing it to decathymidylic acid andthen digesting it with ribonuclease H (Donis-Keller, 1979). This is amuch simpler method of specifically cleaving MDV-1 (+) RNA. Furthermore,cleavage with ribonuclease H leaves the ends of the fragments in thecorrect form for ligation (Berkower et al., 1973). This technique willfacilitate the construction of recombinants from longer heterologoussequences.

The most important use for these recombinant RNAs will be as templatesfor the autocatalytic synthesis of large amounts of RNA in vitro.Recombinant RNAs constructed from appropriate sequences would makeexcellent hybridization probes, since they can be highly labeled andsince blotting with RNA instead of DNA results in lower backgrounds.Recombinant RNAs could be made also from unprocessed gene transcripts,which are difficult to obtain. Once constructed, the recombinants wouldserve as a ready source for the synthesis of additional RNA. These RNAscould then be used as substrates in the isolation of processing enzymesor in studies probing splicing mechanisms (Green et al., 1983).Recombinant RNAs could be made also from eukaryotic mRNAs that aredifficult to obtain. Replication of these recombinants in vitro wouldprovide virtually unlimited quantities of mRNAs for use as templates incell-free translation systems. The presence of the MDV-1 sequences inthe template should not affect protein synthesis, since MDV-1 RNA has noAUG codons or ribosome binding sites, and since the initiation andtermination signals for translation are located within the mRNAsequence. Thus, recombinant mRNAs might provide a novel means forobtaining useful quantities of rare proteins.

Recombinant RNAs could also be used for the analysis and modification ofheterologous sequences. The battery of biological techniques that havebeen developed to study the synthesis of natural templates by Qβreplicase could be applied to recombinant RNAs. For example, thenucleotide sequence of the inserted RNA could be determined directly byreplication of the recombinant in the presence of 3'-deoxyribonucleoside5'-triphosphate chain terminators (Kramer & Mills, 1978; Mills & Kramer,1979). Also recombinant RNAs could be prepared from the genomes ofviruses and viroids. It would then be possible to select mutants ofthese heterologous sequences, possessing particular biologicalproperties, through the evolution in vitro of replicating recombinantRNA populations (Mills et al., 1967; Levisohn & Spiegelman, 1969; Krameret al., 1974). Mutant genomes recovered from these recombinants couldthen be introduced into cells to study their altered biologicalactivity. Thus, the replication of recombinant RNAs constructed fromgenomes and gene transcripts should provide a powerful tool for probingand manipulating genetic information.

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What is claimed is:
 1. A recombinant single-stranded RNA molecule,wherein the sequence required for the initiation of product strandsynthesis is a cytidine-rich 3' terminal sequence.
 2. A recombinantsingle-stranded RNA molecule as in claim 1 which comprises at least oneradiolabeled nucleotide.
 3. A recombinant single-stranded RNA moleculeas in claim 1, wherein the insertion site for the heterologous sequenceof interest is in a loop where viable mutations are known to occur.
 4. Arecombinant single-stranded RNA molecule as in claim 3, wherein theinsertion site is at or near a guanosine residue hypersusceptible tocleavage by ribonuclease T₁.
 5. A recombinant single-stranded RNAmolecule as in claim 1, wherein the MDV-1 RNA is MDV-1 (+) RNA.
 6. Arecombinant single-stranded RNA molecule as in claim 1, wherein theMDV-1 RNA is MDV-1 (-) RNA.
 7. A recombinant single-stranded RNAmolecule as in claim 5, wherein the heterologous sequence of interest isinserted between nucleotides 63 and
 64. 8. A recombinant single-strandedRNA molecule as in claim 7, wherein the inserted heterologous sequenceof interest is decaadenylic acid.
 9. A recombinant, single-stranded RNAmolecule capable of serving as a template in vitro for the synthesis ofa complementary single-stranded RNA molecule by Qβ replicase whichcomprises MDV-1 RNA having the structure shown in FIG. 2 or a mutantthereof and a heterologous RNA sequence inserted into the MDV-1 RNA orthe mutant thereof essentially without changing the secondary ortertiary structure of the MDV-1 RNA, the heterologous sequence ofinterest being inserted between two nucleotides, neither of thenucleotides being (a) hydrogen-bonded to any other nucleotide within theMDV-1 RNA, or (b) present within a Qβ replicase recognition sequence, or(c) present within a sequence for the initiation of product strandsynthesis by Qβ replicase.